Pseudomonas aeruginosa skin-nasopharyngeal colonization in the In-patients: Prevalence, risk factors and antibiotic resistance in tertiary Hospitals in Sana'a city -Yemen.

Pseudomonas aeruginosa is one of most important cause of healthcare-associated infections. This active surveillance cross sectional study was aimed to determine the rate of P.aeruginosa colonization among inpatients at three tertiary hospitals in Sana'a city. In addition, to determine risk factors of colonization and the antibiotic susceptibility of the isolated P.aeruginosa . The study included 327 inpatients. Skin and nasopharyngeal swabs were collected from all participated patients and data were collected by predesigned questionnaire. Standard methods of isolation and identification were used to isolate bacteria in pure culture then identify. Also, antibiotic sensitivity for isolated P.aeruginosa was determined by the disc diffusion method. factors P.aeruginosa of resistant for Amikacin, Ampicillin the of resistant for Gentamicin (35.7%), (11.9%), Tazobactam Ticaracillin calvulanic acid (31%) Colistin sulphat (14.3%). role hospitalization for P.aeruginosa in the hospital and community to tackle this. These data emphasize the need to identify hospitalized patients colonized with P.aeruginosa on admission. Prediction rules or rapid diagnostic testing will help clinicians more appropriately choose empiric antibiotic therapy if subsequent infections occurred. resistance chromosomal genes The results of our study higher rates of resistant of P. (83.3-85.7%) to Amikacin, Ampicillin and This result is different from study carried out at Canada showed significant variable susceptibility pattern with resistance rates to Amikacin, were resistant rates of resistant for isolated


Introduction
Bacterial colonization in general and P.aeruginosa colonization in hospitalized patients are important cause of healthcare-associated infections worldwide 1 . In the United States, it is the 6th most common cause of healthcare-associated infections, accounting for 7.1% of all hospital infections. 2 The choice of empiric antibiotics in the hospital setting is difficult. There needs to be a balance between excessively broad coverage and too narrow coverage. Empiric antibiotic coverage that covers P.aeruginosa but is broader than necessary may lead to the emergence of P.aeruginosa and other intestinal bacteria that are resistant to those broad spectrum antibiotics. In contrast, empiric therapy that does not cover P.aeruginosa may lead to poor outcomes for in-patients eventually found to have P.aeruginosa infection. Improvements in our understanding of which patients require broad-spectrum empiric coverage versus situations in which narrower spectrum agents may be appropriate would be valuable from an antimicrobial stewardship perspective. Knowledge of whether a patient is colonized with P.aeruginosa can be helpful in guiding selection of empiric antibiotics for suspected sepsis in the hospital setting. Colonization with P.aeruginosa is associated with subsequent infection with the same strain of P.aeruginosa 3,4 . The objectives of this active surveillance cross sectional study were as follows: a) Determine the prevalence of P. aeruginosa colonization on Inpatients; b) Determine risk factors for P. aeruginosa colonization; and c) Determine the risk factors of P. aeruginosa colonization.

Subjects and Methods
Site of the study An active surveillance cross sectional study was conducted in in-patients admitted to three tertiary hospitals in Sana'a city: namely: Al-Jumhori hospital, Al-Kuwait hospital and Yemen-Germen hospital, between January 1, 2017, and May 30, 2017. The three hospitals are 1816-bed tertiary care facilities.

Ethical Consideration
Ethical clearance for the study was taken from the Faculty of Medicine and Health Sciences Research Review Committee. A written permission was also taken from the administrative Managers of the Al-Jumhori hospital, Al-Kuwait hospital and Yemen-Germen hospital. Informed Consent was taken from the patients before the questionnaire was filled.

Survey procedure and Laboratory Analysis
Skin and nasopharyngeal swabs collected from inpatients hospitalized in different wards. Clinical and demographic data were collected for all participants' patients. Skin and nasopharyngeal swabs were collected using standard collection techniques, and inoculated on appropriate bacteriological media, including blood agar and Mac-Conkey. Then plates were incubated aerobically at 37°C for 18-24h. Confirmatory tests include production of the blue-green pigment pyocyanin on Cetrimide agar and growth at 42 °C also done. Also, the identification of isolates was made according to standard methods 5 . The identification was made with basic microbiological methods using colony morphology, Gram staining, oxidase, Indole, catalase and coagulase tests etc 5 .

Antimicrobial susceptibility test
Antimicrobial susceptibility test for P. aeruginosa isolates were performed on Mueller-Hinton agar plates by the Kirby-Bauer disc diffusion method 5 . The isolates were tested for their susceptibility against 13 antimicrobial agents that are used in Yemen. Standardized suspension of P. aeruginosa inoculums was compressed to 0.5 McFarland standard turbidity, then inoculated on 3 Muller-Hinton agar plates using a sterile cotton swab by streaking the swab over the entire sterile agar surface 3 times. The plates then allowed drying and antimicrobial discs were placed at the recommended distan C for 24-48 hours before the zones size were record (diameter of inhibition zones was measured and recorded in millimeters with the help of sliding calipers, and an organism was labeled as sensitive or resistance as per CLSI guidelines 5 .

Data analysis
The analysis of data was done by Epi Info version 6 statistical program (CDC, Atlanta, USA), where the chi-q (χ2) v (p) was calculated for the test of significance. In addition, Odd's ratio (OR), 95% confidence interval (95% CI) were added to estimate the risk factors of skin or nasopharyngeal colonization P. aeruginosa on inpatients.

Discussion
In present study , there was a significant rate of skin-nasopharyngeal P.aeruginosa colonization in inpatients equal to 12.8% (table 1). Our result is comparable to that reported from district hospitals in UK 6 . Furthermore our study is similar to that reported previously by Parkins et al. in which hospitalization was a risk factor for bacterial colonization 7 . This association can be explained by that high rate of direct transmission of infectious agents in hospitals, in which this involves a direct body surface-to-body surface contact and physical transfer of microorganisms between a susceptible host and an infected or colonized person, such as when a person turns a patient, gives a patient a bath, or performs other patient-care activities that require direct personal contact. Direct-contact transmission also can occur between two patients, with one serving as the source of the infectious microorganisms and the other as a susceptible host 8 ; or by indirect-contact transmission, which involves contact of a susceptible host with a contaminated intermediate object, usually inanimate, such as contaminated instruments, needles, or dressings, or contaminated gloves that are not changed between patients. In addition, the improper use of saline flush syringes, vials, and bags has been implicated in disease transmission in the US, even when healthcare workers had access to gloves, disposable needles, intravenous devices, and flushes 8 .
Additionally in our study, there was significant association between male patients and skinnasopharyngeal P.aeruginosa colonization in inpatients equal to 2.5 times with CI=1.1-5.5 and PV=0.01, while no significant association was found with female. Our result is similar to that reported previously by several studies in which male sex was a risk factor associated with contracting bacterial colonization in inpatients 1, 7-12 . As well, there was significant association betwe g g ≥50 years and skin-nasopharyngeal P.aeruginosa colonization in inpatients equal to 2.2 times with CI=1.1-4.2 and PV=0.01, while no significant association was found with younger age groups (table 1). This result is parallel to that reported previously by studies in USA and Europe in which older age group was a risk factor associated with contracting bacterial colonization in hospitalized patients 1,10-12 .
When we considered the association between skin-nasopharyngeal P.aeruginosa colonization and type of wards, there was significant associated OR equal to 37 times with CI=16.2-84 and PV<0.0001 with burn wards (table 2). This result is similar to that reported by Lyczak, et al. in which burn wards were associated risk factor with bacterial colonization in inpatients 13 . This association can be explained by that original thermal injury creates a breach in the surface of the skin which make it more susceptible to colonization with micro-organisms, and P. aeruginosa use quorum sensing to induce the production of virulence factors such as proteases, hemolysins, exotoxins A and pyocyanin which help bacteria to colonized 14 . However there was no significant association between ICU wards and skinnasopharyngeal P.aeruginosa colonization (table 2). This result is different from that reported by Harris et al. 6 in which significant association between ICU patients and colonized with P. aeruginosa after admission to hospitals. Our negative result might be due to our small sample size. As soon as we considered the association between skin-nasopharyngeal bacterial colonization and duration of hospitalization, there was significant associated OR equal to 4.0 times with CI=2.0-7.8 and PV<0.001 wi ≥1 z ( ) T which longer period of hospitalization was a hazard factor of bacteria colonization including P.aeruginosa 6,14 . This result can be explained by that contracting of bacteria by patient increase with longer period of exposure to micro-organisms present in hospital. When we considered the association between skin-nasopharyngeal P.aeruginosa colonization and type of underlying diseases, there was no association with malignancy (table 4). This result is dissimilar to all reports in which malignancy was a risk factor for colonization bacteria in hospitalized patients 6 . However, there was significant association between P.aeruginosa colonization and burn with OR= 45, CI=19-105, p<0.001 (table 4). This result is similar to that reported by previous reports [8][9][10][11][12][13] in which burn was risk factor of P.aeruginosa colonization [6][7][8][9][10][11][12][13] . This association can be explained by that original thermal injury creates a breach in the surface of the skin which make it more susceptible to colonization with micro-organisms, and P. aeruginosa use quorum sensing to induce the production of virulence factors such as proteases, hemolysins, exotoxins A and pyocyanin which help bacteria to colonized as stated previously in the discussion of Burn ward 14 .
We carried this study because of the knowledge of P.aeruginosa prevalence and the current antimicrobial profile is necessary in selection of appropriate empirical treatment of these infections and control of P.aeruginosa in hospitals is essential. P.aeruginosa is a common nosocomial pathogen 15 that causes infections with a high mortality rate 16 T , , g ' intrinsically high resistance to many antimicrobials and the development of increased, particularly multidrug resistance in healthcare settings 17 , both of which complicate anti-pseudomonal chemotherapy. Indeed, numerous studies point to a link between multidrug resistance and increased morbidity/ mortality, as well as increased length of hospital stay and increased hospital costs 18 . In our study the isolated P.aeruginosa was completely resistant (100%), to Aztroneome, Ceftriaxone, and Ciprofloxacine. This result is different from that reported in USA in which the rate of Aztroneome, Ceftriaxone, and Ciprofloxacine resistant was not more than 52.2% 19 . Also the incidence of Aztroneome, Ceftriaxone, and Ciprofloxacine resistant in the European countries has been documented by Lambert et al. 16 in which the rates were ranged from 50-72%. These high rates in our study can be x q g [ g , g β-lactamases 20 and aminoglycoside-modifying enzymes 21 via horizontal gene transfer can and do drive antimicrobial/multidrug resistance development in P. aeruginosa, more commonly mutations of chromosomal genes (target site, efflux mutations) 22 . The results of our study showed higher rates of resistant of P. aeruginosa (83.3-85.7%) to Amikacin, Ampicillin sulbactam, and Levofloxacin. This result is different from study carried out at Canada in 2010 which showed significant variable susceptibility pattern with lower resistance rates to Amikacin, (25%), Ampicillin sulbactam (35%), and Levofloxacin (25%). There were low resistant rates of resistant for our isolated P.aeruginosa for Gentamicin (35.7%), Imipenem (11.9%), Pipracillin Tazobactam (11.9%), and Ticaracillin calvulanic acid (31%). The current results are similar to that reported from USA in which resistance rates to these antibiotics were not exciding 30%. 17 Owing to the increased prevalence of multidrug-resistant P. aeruginosa, " " k x ( x B ) k v , with earlier issues surrounding nephrotoxicity largely dealt with 23,24 . The rate in our study for Colistin sulphat resistant was 14.3%. This rate is similar to that reported from Russia and Western Europe. 23,24 Conclusion The study have been highlighted the role of hospitalization as a significant risk for P.aeruginosa colonization; concerted and coordinated efforts are required both in the hospital and community to tackle this. Elimination of health care-associated infections is a priority of the Ministry of Health and Population. Continued improvements in patient safety depend on maintaining a comprehensive understanding of the epidemiology of health care-associated infections. Currently, no single Yemen surveillance study can provide estimates of the burden of all types of such infections across hospital care patient populations. Effective antimicrobial activity as well as cost effectiveness should be considered in drugs prescribed for P.aeruginosa infections. Oral dosing options for antibiotics can allow earlier discharge of hospitalized patients and minimize the chances of P.aeruginosa multiresistant emergence. Good hospital infection control measures prove to be the main stay against these infections because antibiotics can never be an effective alternative to good medical practice. Limitation This study has a number of limitations. Some data is missing and the numbers are small. Only skin and nasopharyngeal specimens were collected. In the protocol of the study rectal colonization was concerned, but most patients were refused to give rectal swab so no more than skin and nasopharyngeal specimens were collected. No molecular studies were used to categories samples further, in order to determine cross resistance or resistance mechanisms. Nevertheless, we believe this study adds further information to the epidemiology of a significant pathogen in the hospital setting in Sana'a city. In addition, although we are confident that the studied hospitals are representative of hospitals within Sana'a city, they may not be representative of all Yemen hospitals.